Genome sequencing of Prototheca
zopfii genotypes 1 and 2 provides
evidence of a severe reduction in
organellar genomes
Marco Severgnini
1, Barbara Lazzari
2,3, Emanuele Capra
3, Stefania Chessa
3, Mario Luini
4,
Roberta Bordoni
1, Bianca Castiglioni
3, Matteo Ricchi
5& Paola Cremonesi
3Prototheca zopfii (P. zopfii, class Trebouxiophyceae, order Chlorellales, family Chlorellaceae), a
non-photosynthetic predominantly free-living unicellular alga, is one of the few pathogens belonging to the plant kingdom. This alga can affect many vertebrate hosts, sustaining systemic infections and diseases such as mastitis in cows. The aim of our work was to sequence and assemble the P.
zopfii genotype 1 and genotype 2 mitochondrial and plastid genomes. Remarkably, the P. zopfii
mitochondrial (38 Kb) and plastid (28 Kb) genomes are models of compaction and the smallest known in the Trebouxiophyceae. As expected, the P. zopfii genotype 1 and 2 plastid genomes lack all the genes involved in photosynthesis, but, surprisingly, they also lack those coding for RNA polymerases. Our results showed that plastid genes are actively transcribed in P. zopfii, which suggests that the missing RNA polymerases are substituted by nuclear-encoded paralogs. The simplified architecture and highly-reduced gene complement of the P. zopfii mitochondrial and plastid genomes are closer to those of
P. stagnora and the achlorophyllous obligate parasite Helicosporidium than to those of P. wickerhamii
or P. cutis. This similarity is also supported by maximum likelihood phylogenetic analyses inferences. Overall, the P. zopfii sequences reported here, which include nuclear genome drafts for both genotypes, will help provide both a deeper understanding of the evolution of Prototheca spp. and insights into the corresponding host/pathogen interactions.
Organisms belonging to the genus Prototheca are achlorophyllous algae widespread in the environment. The genus is classified within the class of Trebouxiophyceae, order Chlorellales and family Chlorellaceae, and histori-cally encompasses six species: P. stagnora, P. ulmea, P. wickerhamii, P. blaschkeae, P. zopfii and P. cutis1–3. A seventh
species, P. miyajii, has very recently been isolated in a patient with systemic protothecosis and classified as a sep-arate species due to some genetic and phenotypical differences from P. wickerhamii4. Finally, an eighth species, P.
moriformis, is not currently considered a species per se because of its biochemical/genetic resemblance to P. zopfii and because of its high intraspecific heterogeneity2,5.
All Prototheca species have forfeited their photosynthetic capabilities, and, consequently, their ability to har-vest energy from light and fix carbon, having undergone an evolutionary transition from autotrophy to hetero-trophy3, favoured also by the ability of some species to sustain infectious diseases in both humans and animals6,7.
P. wickerhamii, P. cutis and P. blaschkeae, in particular, have been associated with human diseases, especially in the presence of impaired immunological-cellular systems1,7,8. Nevertheless, P. wickerhamii, P. blaschkeae and P.
zopfii can also infect animals, especially dogs and cows6,9,10. Among them, P. blaschkeae and P. zopfii, are the most
important species in the veterinary field because of their ability to sustain bovine mastitis11–13. P. zopfii can be
further divided into two genotypes, namely genotype 1 and 2, both reported as pathogenic for humans14, whereas
genotype 2 is the most isolated Prototheca in bovine mastitis outbreaks worldwide11,12,15–18.
1Institute of Biomedical Technologies, National Research Council (ITB-CNR), Segrate, Milan, Italy. 2PTP-Science
Park, Lodi, Italy. 3Institute of Agricultural Biology and Biotechnology, National Research Council (IBBA-CNR), Lodi,
Italy. 4Lombardy and Emilia Romagna Experimental Zootechnic Institute (IZSLER), Lodi, Italy. 5Lombardy and
Emilia Romagna Experimental Zootechnic Institute (IZSLER), Piacenza, Italy. Marco Severgnini, Barbara Lazzari and Emanuele Capra contributed equally. Correspondence and requests for materials should be addressed to M.R. (email:
[email protected]) Received: 9 February 2018
Accepted: 12 September 2018 Published: xx xx xxxx
The sequences of Prototheca species currently available in public databases are those of the 18S rDNA (small subunit of rDNA, SSU) and 28S rDNA (large subunit of rDNA, LSU)2,19, and those of the Internal Transcribed
Spacer regions (ITS), as well as some mitochondrial and plastid genomes. Notably, this information is not avail-able for all species and, full-length sequences are very often missing12,20,21: complete sequences of the organellar
DNA are currently only available for P. wickerhamii (both mitochondrion and plastid)22,23, P. cutis and P. stagnora
(plastid only)24.
In this paper, we present the complete and manually annotated genomes of mitochondria and plastids of P. zopfii genotypes 1 and 2, and the first draft assembly of the whole nuclear genomes of both P. zopfii subspecies. Our work gives, for the first time, a representative overview of the extreme reduction which occurs within the mitochondrial and plastid genomes of these algae and provides basic information for further investigations.
Materials and Methods
Strains and culture conditions.
P. zopfii genotype 1 (SAG 2063) was obtained from the Culture Collection of Algae at Göttingen University (“Sammlung von Algenkulturen der Universität Göttingen”, SAG, Göttingen, Germany). P. zopfii genotype 2 (SAG 2021) was kindly donated by Dr. Jagielski, University of Warsaw (Poland). The strains were aerobically sub-cultured on Sabouraud Dextrose Agar plates for 48–72 h at 30 °C until DNA isolation was carried out.Isolation of DNA and RNA.
Genomic DNA and RNA were extracted starting from approximately one g of P. zopfii genotype 1 and genotype 2 resuspended in 20 ml wash solution (0.6 M Sucrose, 20 mM Tris, 20 mM MgCl2 and 1 mM DTT, pH 7.5) and centrifuged at 2200 g for 2 min at 4 °C.For DNA, washed pellets were resuspended in 500 μl of TRIS EDTA lysis buffer (10 mM Tris-HCl, 10 mM EDTA, 250 mM NaCl, pH 8), supplemented with 25 µl of proteinase K (20 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA), 25 µl of SDS 10% and incubated at 56 °C for 2 h. Next, 25 µl of RNaseA (20 mg/ml) (Sigma-Aldrich) were added and the suspensions were incubated at 56 °C for 30 min. DNA was extracted using an equal volume of 1:1 (v/v) phenol:chloroform and precipitated with one volume of cold isopropanol. DNA was rinsed with 70% (v/v) cold ethanol, air dried, resuspended in 30 µl of ultrapure water and stored at −20 °C until use. DNA concentration and quality were estimated by PicoGreen (Thermo Fisher, Waltham, MA, USA) and by agarose gel electrophoresis.
Total RNA was extracted from washed pellets with TRIzol (Invitrogen, Carlsbad, CA, USA) and purified by NucleoSpin miRNA kit (Macherey-Nagel, Duren, Germany), following the manufacturer’s protocol, in combina-tion with TRIzol lysis. RNA concentracombina-tion (ng/µl) and quality RNA Integrity Number (RIN) were determined by Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). RNA extracts were stored at −80 °C until use.
DNA and RNA library preparation and sequencing.
P. zopfii DNA libraries for Illumina sequencing (paired-end and mate-pair sequencing) (Illumina, San Diego, CA, USA) were prepared and sequenced on an Illumina MiSeq instrument, following the manufacturer’s instructions as detailed in Supplementary Table 1. DNA library preparation for GS-FLX sequencing was performed using the GS FLX Titanium Rapid Library Preparation Kit (Roche, Basel, Switzerland) as follows: 1 μg DNA for each P. zopfii strain was used in the preparation of shot-gun libraries by genomic DNA fragmentation by nebulization and ligation to specific adapters. According to the manufacturer’s instructions, libraries were subjected to clonal amplification by emulsion PCR reaction, recovered by isopropanol breaking and enriched for positive reaction beads. Each library was separately loaded onto one region of the GS-FLX PicoTiter Plate and sequenced according to the 454 GS-FLX Titanium XL protocol.One μg of RNA was used for libraries construction using TruSeq
®
RNA Sample Preparation v2 Kit (Illumina), according to the manufacturer’s instructions using poly(A) enrichment and sequenced on a 2 × 101-cycles Hiseq 2000 run (Illumina).De novo genome assembly.
The whole genomic sequence collections - composed of Illumina paired-end and mate-pair sequences, plus GS-FLX reads - from P. zopfii genotype 1 and 2 were assembled with SPAdes25 inread error correction and assembling mode. Six K values, ranging from K21 to K127, were automatically selected by the algorithm based on the read length and dataset type. CAP326 (-p 96 -o 500) was run on the resulting
scaf-folds to further assemble contiguous regions. Illumina reads were mapped back to scafscaf-folds by means of bwa mem (v. 0.7.1027), keeping only those having ≤2 mismatches with the reference and Pilon (v 1.22)28 in assembly
improvement mode was employed in order to correct substitutions and short indels.
Organelles assembly.
Among the assembled scaffolds, sequences of mitochondrion and plastid were detected by homology with a set of 322 mitochondrial and 605 plastid/chloroplast sequences from related spe-cies (i.e.: green algae) and circularized, giving rise to full circular sequences for both organelles in both P. zopfii genotypes. Assembly of the mitochondrial and plastid genomes of P. zopfii was independently confirmed by a custom assembly strategy that, starting from “seeds”, implements an iterative procedure aimed at finding reads partially overlapping with the seed and assembling them with the original contig. Then, overlaps among the new contigs were used to generate a “supercontig”. Further alignment-assembly-extension steps were performed on each side of the supercontig until an overlap between the 5′ and 3′ ends of the sequence was found, meaning that the whole circular genome had been covered. A full description of this custom assembly procedure is available in Supplementary Methods.Nuclear genome annotation.
Gene prediction in nuclear genomes was performed with Augustus29using Chlorella variabilis as reference species. A double annotation procedure was carried out on the predicted protein models, employing both BLAST30 (-evalue 1e−10) versus the UniProtKB database (http://www.uniprot.
performed between the annotated protein datasets of the two P. zopfii genotypes. Annotation of proteins iden-tified as putative DNA-directed RNA polymerases (NEPs) was double-checked by BLASTp against the NCBI’s own non-redundant (“nr”) database. Presence of a chloroplast transit peptide (cTP) in genes annotated as NEPs was assessed by using several prediction tools available on the web: PProwler32, PredAlgo33, TargetP (v1.1)34,
and predSL35.
Organelles annotation.
Organelles were annotated using the DOGMA webserver36. Annotation wasman-ually refined, relying on similarities obtained by BLAST versus the organellar genes of the closest known relatives of P. zopfii (i.e.: C. variabilis, Auxenochlorella protothecoides, Helicosporidium sp. and P. wickerhamii). Transfer RNA (tRNA) and ribosomal RNA (rRNA) sequences were determined by BLASTn alignment, whereas coding DNA sequences (CDS) positions were refined on the basis of protein-protein (BLASTp) matches. Circular rep-resentations of the mitochondria and plastids were drawn using Gview37. Comparison of CDS among species was
performed by BLASTp.
Phylogenetic analysis.
The protein sequences inferred from nine conserved plastid ribosomal genes (RPL2, RPL5, RPL14, RPL16, RPL20, RPS8, RPS11, RPS12, RPS14) were retrieved from P. zopfii genotype 1 and 2, from related species (i.e.: C. variabilis, A. protothecoides, Helicosporidium sp., P. wickerhamii, P. cutis and P. stagnora), and from other Trebouxiophyceae species available at NCBI, for a total of 40 species. Only ribosomal proteins whose sequences were available for each considered species were taken into account. Sequences were concatenated and aligned to produce a super-alignment with Clustal-Omega38, which was manually inspectedand used to infer a Maximum Likelihood phylogenetic tree with the program PhyML39 using four substitution
rate categories, the cpREV substitution model, estimated gamma shape parameter, 1000 bootstraps, and core Trebouxiophyceae as outgroup.
RNA-Seq data analysis.
RNA-Seq reads from P. zopfii 1 and 2 were mapped to the respective draft assem-blies with STAR aligner (v 2.5.3a)40. Alignments were filtered retaining, for each genotype, only reads aligningfor ≥80% of their length and having ≤2 mismatches. The number of reads mapping within the predicted gene models was assessed with BEDtools (v 2.26.0)41, requiring a read to map for at least 90% of its length within a gene
to be counted. Counts were then converted to reads-per-kilobase-per-million (RPKM) values.
Size (nt) Number Scaffolds %GC Total features CDS tRNA rRNA Number introns (size) GenBank Accession
Mitochondrion C. variabilis 78,500 1 28.2 62 32 27 3 6 (5,482) NC_025413.1 A. protothecoides 57,274 1 28.7 70 39(+2c) 26 3 7 (6,589) NC_026009.1 Helicosporidium sp. 49,343 1 25.6 65 37 25 3 2 (8,208) NC_017841.1 P. wickerhamii 55,328 1 25.8 65 36 26 3 5 (4,709) NC_001613.1 P. zopfii gen. 1 38,164 1 28.7 62 33 26 3 0 (0) MF197533.1 P. zopfii gen. 2 39,222 1 28.7 63 34 26 3 1 (776) MF197534.1 Plastid C. variabilis 124,793 1 34.0 112 79 30 3 3 (1,657) NC_015359.1 A. protothecoides 84,576 1 30.8 109 76 30 3 0 (0) NC_023775.1 Helicosporidium sp. 37,454 1 26.9 54 26 25 3 1 (486) NC_008100.1 P. stagnora 48,188 1 25.7 56 25 (+3d) 25 3 0 (0) AP018372.1 P. cutis 51,673 1 29.7 72 40 29 3 0 (0) AP018373.1 P. wickerhamii 55,636 1 31.1 70 40 27 3 0 (0) KJ001761.1 P. zopfii gen. 1 28,698 1 27.0 47 19 25 3 0 (0) MF197535.1 P. zopfii gen. 2 28,638 1 26.8 47 19 25 3 0 (0) MF197536.1 Nuclear
C. variabilis 42.2 M 414 67.1 9,780 9,780 — — n.a. ADIC00000000.1
A. protothecoides 22.9 M–32.7 Ma 113 62.8 7,016 7,014 — 2 n.a. APJO00000000.1
Helicosporidium sp. 12.4 M 5,666 61.7 6,033 6,033 — — n.a. AYPS00000000.1
P. stagnora 16.9 M 27 71.4 7,041 7,041 — — n.a. BCJY00000000.1
P. cutis 20.0 M 29 60.3 6,884 6,884 — — n.a. BCIH01000000.1
P. wickerhamii ~29.0 Mb 2,860b n.a. n.a. n.a. n.a. n.a. n.a. —
P. zopfii gen. 1 ~26.5 M 6,956 67.3 6,884 6,884 — — n.a. PEIA01000000
P. zopfii gen. 2 ~24.7M 4,555 73.5 6,381 6,381 — — n.a. PGFX00000000
Table 1. Organelles and nuclear genome annotation statistics. For each organism, the nuclear or organellar DNA size, the number of scaffolds in the assembly, the percentage of GC content (%GC), the number of introns and size, the number of genes (Total features), subdivided into: coding sequences (CDS), the number of transfer RNAs (tRNA), and of ribosomal RNAs (rRNA) are reported; last column reports NCBI GenBank Accession number for the assemblies. aOriginal WGS sequencing (BioProject PRJNA182710, year: 2014) covers 21,856,191 bp, whereas a more recent sequencing (PRJNA362700, year: 2017) estimates a total length of 32,730,026 bp. bAs reported in51. cA. protothecoides mitochondrion also includes 2 pseudogenes. dP. stagnora plastid also includes 3 ORFs of unknown function.
Results
The genomes of P. zopfii genotype 1 and 2 were sequenced using a combination of different approaches (Supplementary Table 1) resulting in 45,166,626 and 66,488,185 total reads, respectively.
Sequence assembly.
Sequence assembly yielded a nuclear genome of 26,448,891 and 24,744,895 bp for P. zopfii genotype 1 and genotype 2, respectively; organelles were very small: mitochondria being 38,164 and 39,222 bp for genotypes 1 and 2, respectively, whereas plastids being 28,698 and 28,686 bp, for genotype 1 and 2 respectively (Table 1).Mitochondrial structure and annotation.
The mitochondrial genomes of both P. zopfii genotype 1 and 2 are sized at about 38–39 Kb (Fig. 1A, Supplementary Fig. 1A). They are extremely compact, with only about 32% of non-coding DNA and are characterized by a substantial loss of any intron-exon structure in their genes. Only P. zopfii genotype 2 shows a single intron (length: 777 bp) in the long ribosomal subunit (lrn) gene, whereas the other species belonging to the Chlorellales (i.e.: C. variabilis, A. protothecoides, Helicosporidium sp., and P. wick-erhamii) display a more complex structure, with intron length reaching 4,000–8,000 bp. A putative LAGLIDADG homing endonuclease, a class of restriction enzymes directly involved in the DNA cutting process42, is encodedwithin the intron of lrn gene.
The annotation revealed a number of coding genes for both P. zopfii species similar to those observed in other Chlorellales species. All the genes encoding for cytochrome units, NADH dehydrogenase, ATP synthases and ribosomal proteins, as well as all the tRNAs and the ribosomal units, have a conserved structure (Table 1, Fig. 2A). As expected, mitochondrial protein similarity between P. zopfii genotype 1 and 2 is above 90%, while similarity with Helicosporidium sp., C. variabilis, A. protothecoides and P. wickerhamii is around 60%.
Plastid structure and annotation.
The structure of the plastid genomes of P. zopfii genotype 1 and 2 (Fig. 1B, Supplementary Fig. 1B) is very similar to that of other non-photosynthetic algae belonging to the Trebouxiophyceae class, being extremely compact. The genomes are sized only about 28.7 Kb for both genotypes and are the shortest plastid genomes within their class. Both P. zopfii genotypes possess only 19 CDS, and as a result are simpler than those of Helicosporidium sp. (26 CDS), P. wickerhamii, P. cutis (both possessing 40 CDS) and P. stagnora (28 CDS). On the other hand, the tRNAs and rRNAs are conserved among all these species and other photosynthetic algae (i.e.: C. variabilis and A. protothecoides) (Table 1, Fig. 2B). Plastid protein similar-ity between genotype 1 and 2 is about 85% while similarsimilar-ity with other organisms is around 41–43%, with the best match being with P. stagnora (i.e.: 49.9% and 49.5% similarity to P. zopfii 1 and 2, respectively) (Fig. 3A). Comparison with other related organisms shows that the plastid genomes of P. zopfii genotype 1 and 2, like those of Helicosporidium sp., P. wickerhamii, P. cutis and P. stagnora, lack the genes associated with photosynthesis (photosystem I and II, chlorophyll biosynthesis and cytochrome components), whereas ATP synthase genes were Figure 1. P. zopfii genotype 2 mitochondrion and plastid circular plot. Circular plots depicting the annotation of P. zopfii genotype 2 mitochondrion (A) and plastid (B). Gene annotation is reported on the outermost circle of the plot; CDS are in blue, tRNA are in green and rRNA are in red. Innermost circles represent gene orientation, GC content and skew. Other rings report the extent and the % identity of the plastid features with those of proximal organisms (C. variabilis, A. protothecoides, Helicosporidium sp., P. wickerhamii plus P. cutis and P. stagnora for plastid only) and with P. zopfii genotype 1. Transparency is proportional to the degree of identity between P. zopfii genotype 2 and each reference genome; no transparency indicates 100% identity. % identity was calculated on the basis of BLASTn (for tRNA and rRNA) and BLASTp (for CDS) matches with the corresponding features on the reference plastid genome.maintained only in P. wickerhamii and P. cutis. Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL, or RuBisCO) is absent in P. zopfii, as well as in all other Prototheca spp. and in Helicosporidium sp. Moreover, differently to the other Trebouxiophyceae, P. zopfii also lacks all plastid-encoded RNA polymerases (i.e.: rpoA, rpoB, rpoC1 and rpoC2) (Table 2, Supplementary Table 2). Our transcriptome analysis demonstrated that most plastid genes encoding rRNA and proteins were expressed in P. zopfii (Supplementary Fig. 2A), although three of them (i.e.: rps4, rps7, rpl20) had low RPKM values, suggesting that nuclear-encoded counterparts could compen-sate for the loss of plastid-encoded RNA polymerases.
Both P. zopfii genotypes plastids contain the genes of most of the ribosomal proteins, with the exception of RPL12, RPL19, RPL23, RPL32, RPS2, RPS18 and RPS9. The same proteins, with the exception of RPL19 and RPL32, are also absent in P. stagnora, which, apparently, is the most similar organism. The phylogenetic tree inferred from the super-alignment of 9 shared ribosomal proteins confirms that Helicosporidium sp., P. stagnora and P. zopfii are closely related genera, whereas P. wickerhamii is more distant, closer to A. protothecoides than to other Prototheca species (Fig. 3B).
Nuclear genome annotation.
From the genome assembly procedure, 6,956 and 4,555 scaffolds with lengths >1 Kb representing the nuclear genome were obtained for P. zopfii genotype 1 and 2, respectively, indi-cating a genome size of about 26.5 Mbp and 24.7 Mbp for genotypes 1 and 2, respectively, a size between those of Helicosporidium sp. (12.4 Mb) and C. variabilis (46.2 Mb) and comparable to the genome of A. protothecoides (~23 Mb) and P. wickerhamii (~29 Mb) (Table 1).The maximum scaffold length was 97,625 and 57,068 bp, with 1,289 and 1,708 contigs exceeding 5 Kb in length and a N50 value of 6,686 and 7,940 for P. zopfii genotypes 1 and 2, respectively.
Figure 2. Multi alignment of P. zopfii mitochondrion and plastid sequences. Gene order comparison between the mitochondrial (A) and plastid (B) sequences of P. zopfii and other members of the Trebouxiophyceae class. Organisms are ordered by descending organelle size.
Augustus gene prediction led to the individuation of 6,884 and 6,381 gene models for the two genotypes. 56.5% and 62.0% of P. zopfii genotype 1 gene models were annotated versus UniProtKB and InterPro databases, respectively, while the corresponding percentages for genotype 2 were 59.4% and 67.2%. The main features of the nuclear genome, such as gene number and coding density, were consistent for both genotypes, and similar to their counterparts in A. protothecoides genome (i.e.: gene density: 0.26 genes/Kb and 0.32 genes/Kb, for both P. zopfii genotypes and A. protothecoides, respectively). On the contrary, the average exon and intron sizes were higher compared to those previously observed in related species (Supplementary Table 3). BLAST comparison of the predicted proteins of the two P. zopfii genotypes resulted in a set of 6,134 common entities, representing a core set of homologous proteins conserved in the two genotypes. Among the predicted genes and transcripts, we found evidence of the presence and expression of nuclear-encoded polymerases (NEPs) (Supplementary Data 1 and 2). P. zopfii genotype 1 and 2 possess 21 and 19 genes annotated as NEPs, respectively, and both showed a RNA-Seq signal indicating active transcription of many of them (Supplementary Fig. 2B,C). Prediction of target peptides highlighted at least one NEP per genotype as a high-confidence candidate for containing a chloroplast transit pep-tide (cTP) (genes g5108 and g2780 for genotypes 1 and 2, respectively, for which all the four prediction software employed were concordant); moreover, PredAlgo also suggested two more genes (g3914 and g4216 for genotypes 1 and 2, respectively) to be plastid-directed NEPs. In addition to that, we found evidence of some mitochondrial targeting peptides (mTPs) in more gene models (Supplementary Table 4).
Figure 3. Phylogenetic analysis of P. zopfii. (A) Maximum Likelihood (ML) tree inferred from the super-alignment of 9 plastid ribosomal proteins (i.e.: RPL2, RPL5, RPL14, RPL16, RPL20, RPS8, RPS11, RPS12, and RPS14). Bootstrap values are indicated above the lines. Core Trebouxiophyceae was used as outgroup. Among Chlorellales, (dark green vertical bar) non-photosynthetic species are highlighted in blue; tree lengths for this group are not drawn to scale. (B) Heatmap representing the pairwise average percentage of identity between CDS of 8 organisms belonging to Chlorellales (P. zopfii and its closest relatives).
Functiona Presentb Absentc
Transcription (plastid-encoded RNA polymerase) — rpoA, rpoB, rpoC1, rpoC2
Translation — tufA
Ribosomal proteins Small subunit rps3, rps4, rps7, rps8, rps9, rps11, rps12, rps14, rps19 rps2, rps18
Large subunit rpl2, rpl5, rpl14, rpl16, rpl20, rpl36 rpl12, rpl19, rpl23, rpl32
Photosynthesis
ATP synthase — atpA, atpB, atpE, atpF, atpH, atpI Photosystem I — psaA, psaB, psaC, psaI, psaJ, psaM Photosystem II — psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM,
psbN, psbT, psbZ Cytochromecomplex — petA, petB, petD, petG, petL
Metabolism accD, cysT rbcL, cysA
Chlorophyll biosynthesis — chlB, chlI, chlL, chlN
Protein quality control ftsH clpP
Assembly, membrane insertion — ccsA, secG
Table 2. Comparison of genes of the C. variabilis chloroplast with genes of the P. zopfii and P. stagnora plastids. Genes are grouped according to function. aFunction and names of chloroplast genes follow the categorization and nomenclature reported in52. bGenes present in P. zopfii. Genes present in P. zopfii genotype 1 and 2 but absent in P. stagnora plastid are in boldface. cGenes absent in both P. zopfii and P. stagnora. Genes present in P.
Discussion
In this paper, we describe the complete, manually annotated, circular sequence of both mitochondrial and plastid organellar DNA of P. zopfii genotype 1 and genotype 2, as well as a first draft of the complete genome, by whole genome shotgun sequencing.
Structure of the mitochondrial genomes of both P. zopfii genotypes was revealed to be smaller in size and extremely condensed when compared to that of some related organisms, i.e.: P. wickerhamii22 and Helicosporidium
sp.43, but similarly functional, with the size reduction mostly due to the lack of intron-exon structures.
An extremely compact and simplified structure was also observed in P. zopfii plastid genomes, which showed a substantially reduced size (about 28.7 Kb), smaller than those of all other algae belonging to the class of Trebouxiophyceae. As previously observed in the genomes of non-photosynthetic algae belonging to this class, P. zopfii plastids lack all the genes for the synthesis of the proteins involved in the photosynthesis process23,44, and
for the RuBisCO large subunit. Fundamental plastid-related functions, however, seem to have been preserved, as indicated by the presence of a RNA-Seq signal on the genes of plastid-encoded ribosomal proteins. The low expression of some of them, however, cannot preclude their presence in the genome as pseudogenes in P. zopfii. However, further experiments should be carried on in order to confirm this observation. More interestingly, the entire set of rpo genes (i.e. rpoA, rpoB, rpoC1 and rpoC2), which codify for the plastid-encoded RNA poly-merases (PEPs), was lost in P. zopfii, an unprecedented observation within this class of algae. Loss of PEPs was previously reported for other non-photosynthetic parasitic plants, such as Cuscuta obtusiflora45 and Rhizanthella
gardneri46, but not in apicomplexan and algae: plastid genomes of Plasmodium falciparum47, A. protothecoides, P.
wickerhamii23, P. cutis, P. stagnora24 and Helicosporidium sp.44 have all retained the complete set of rpo genes. We
found no evidence of PEP sequences either in plastid assemblies or in the nuclear draft genomes, whereas nuclear genome contigs contained evidence of 21 and 19 DNA-driven, nuclear-encoded polymerases (NEPs) for P. zopfii genotype 1 and 2, respectively, and at least one of them, per genotype, was predicted to contain plastid-targeting signal peptides. It is therefore possible that P. zopfii codes for other NEPs able to target the plastid, making it pos-sible to transcribe its genetic information.
As previously suggested23,24,48,49, considering the degree of similarity between the few structural genes
pre-served in its plastid genome and the evidence from the phylogenetic analysis, P. zopfii seems to be more closely related to P. stagnora and to Helicosporidium sp., rather than to P. wickerhamii or P. cutis. Moreover, it is notewor-thy that P. wickerhamii appears not to be closely related to P. zopfii, but instead to A. protothecoides, strengthening the evidence that P. wickerhamii is only loosely related to other Prototheca spp, as previously revealed by plastid genome comparison23 and supporting the proposal of either moving P. wickerhamii into Auxenochlorella genus
or creating a new genus48.
Nuclear genome assemblies of P. zopfii genotype 1 and 2 had a size estimated at about 25–26 Mb for both, consistent with that reported in a previous work50. Although further studies are certainly needed to elucidate the
structure of nuclear genomes of P. zopfii, this work adds information to the growing body of genome resources for the plant kingdom, being, although a preliminary draft, the first report of the assembly of nuclear DNA of P. zopfii. In conclusion, we believe that the information reported herein will be important for the understanding of the evolution and genomic organization of Prototheca spp., with a particular focus on the progressive loss of functions of plastids in the shift from autotrophic, photosynthetic, to obligate, heterotrophic, parasitic algae.
Data Availability
The complete genome sequencing project has been registered in the NCBI BioProject portal (https://www.ncbi.
nlm.nih.gov/bioproject/) under the accession number PRJNA388740. Raw DNA sequence reads for P. zopfii
gen-otypes 1 and 2 have been deposited into the NCBI Short-Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra/) under the accession numbers SRR6319956-SRR6319964. RNA-seq reads are saved under the accession numbers SRR7091517-SRR7091518. Full sequences of mitochondria and plastids are available in GenBank, under acces-sions MF197533, MF197534, MF197535, and MF197536. The Whole Genome Shotgun project has been deposit-ed (as non-annotatdeposit-ed contigs) at DDBJ/ENA/GenBank under the accessions PEIA00000000 and PGFX00000000. The versions described in this paper are PEIA01000000 and PGFX01000000. Sequences annotated as nuclear-en-coded polymerases (NEPs) are available as amino acid FASTA files in Supplementary Data 1 and 2.
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Acknowledgements
We thank Dr. Tomasz Jagielski from the University of Warsaw (Poland) for providing P. zopfii genotype 2 strain. We also thank Mr. Bryan Manchi for his valuable revision of the English text.
Author Contributions
M.S. and B.L. assembled and annotated the genome, conducted analyses and wrote the paper. E.C. prepared P. zopfii genome samples, performed genomic analyses and wrote the paper. R.B. prepared and sequenced P. zopfii samples on GS-FLX platform. M.R. helped during the conception of the study, provided P. zopfii and wrote the paper. M.L. provided P. zopfii and helped draft the manuscript. S.C. sequenced P. zopfii samples on Illumina MiSeq instrument and helped draft the manuscript. B.C. conceived the study and helped draft the manuscript. P.C. conceived the study, helped in preparing P. zopfii genome samples, sequenced P. zopfii samples on Illumina MiSeq instrument and wrote the paper. All authors read and approved the final manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-32992-0. Competing Interests: The authors declare no competing interests.
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